Recent studies from this laboratory have suggested that in both atherosclerosis and hypertension, the function of the endothelial nitric oxide synthase (eNOS) is altered such that it produces superoxide rather than nitric oxide (NO). This phenomenon has been referred to as eNOS "uncoupling". Preliminary studies indicate that peroxynitrite, formed by the reaction between superoxide and nitric oxide, oxidizes tetrahydrobiopterin (BH4), a critical co-factor of eNOS, and that this leads to uncoupling of the enzyme in vivo. Under normal conditions a major source of reactive oxygen species in the endothelium is the NADPH oxidase which by producing superoxide could participate in peroxynitrite production. The project is to test the hypothesis that hypertension may initially stimulate NADPH oxidase to produce "kindling radicals" (in particular peroxynitrite), that in turn oxidize tetrahydrobiopterin and uncouple eNOS. In aim 1, DOCA-salt hypertension will be created in gp91phox mice. These animals lack NADPH oxidase activity in the endothelium and eNOS will not be uncoupled. In aim 2, to prevent eNOS uncoupling, DOCA-salt hypertensive rats will be treated with ebselen, a potent peroxynitrite scavenger and oral BH4, either alone or in combination. The effect of these interventions on vascular and endothelial BH4 levels will be determined. In aim 3, the role of endothelial and eNOS-derived hydrogen peroxide on vascular hypertrophy will be determined using DOCA-salt hypertension mice that overexpress catalase in the endothelium and are deficient in eNOS. Aim 4 is to define the enzymatic source involved in oscillatory shear-induced superoxide production by cultured endothelial cells using electron spin resonance.